DISCUSSION

Molecular prevalence of T. orientalis in cattle and ticks

Information regarding T. orientalis infection in cattle remains limited in Iraq [24], although recent studies conducted in Asia [30, 31], Africa [32, 33], the Americas [34], and Europe [35, 36] have demonstrated that this tick-borne parasite is associated with considerable morbidity, production losses, and, in some cases, mortality in cattle. In the present study, the molecular prevalence of T. orientalis was 12.35% in cattle and 9.41% in their naturally infesting ticks. These findings provide the first simultaneous molecular evidence of T. orientalis infection in cattle and associated ticks from Wasit Province, Iraq.

Compared with previous reports, the prevalence observed in this study was higher than those reported from Ethiopia (2.2%) [37], Turkey (5.6%) [38], and Egypt (8.8%) [39], but was comparable with reports from Vietnam (13.8%) [40] and Pakistan (15.0%) [41]. Lower prevalence than the present study has also been reported in northeastern Thailand (30.1%) [42], Kazakhstan (33.3%) [43], China (14.27%–36.5%) [30, 44], Malaysia (49.76%) [45], Japan (10%–64.8%) [46], and India (83.3%) [31]. Such wide geographical variation in the prevalence of bovine theileriosis is likely attributable to differences in climatic conditions, tick vector abundance, cattle management systems, host immunity, diagnostic approaches, and the genetic diversity of circulating Theileria strains [47].

Environmental factors strongly influence tick ecology, with warm and humid climates generally favoring tick survival and transmission, thereby increasing disease prevalence [48]. In addition, farm management practices, including grazing systems, biosecurity measures, and nutritional status, may alter host susceptibility by increasing physiological stress [49]. Farms managed as small-scale or sideline enterprises often have limited resources for tick control, hygiene, and routine veterinary surveillance, which may further increase the risk of blood parasite transmission [50]. Moreover, genetic variation among Theileria species, including the emergence of strains with different levels of virulence or antimicrobial resistance, may further contribute to regional differences in disease epidemiology [51]. Variability in reported prevalence may also reflect differences in molecular diagnostic procedures, including pre-analytical sample handling, analytical protocols, and biological factors affecting parasite detection.

Association of T. orientalis infection with host-related risk factors

The present study demonstrated that T. orientalis infection was significantly associated with animal age. The highest prevalence was observed in cattle aged 1–3 years and >3–6 years, whereas the highest risk of infection was detected in cattle aged >3–6 years. Likewise, the prevalence of PCR-positive tick samples was greatest among cattle aged 1–3 years. Interpretation of age-related susceptibility should be approached with caution because previous studies have not consistently distinguished between epidemic and endemic disease conditions [6, 52–54].

Previous investigations have reported inconsistent findings regarding the influence of age on T. orientalis infection. In Australia, T. orientalis was detected microscopically in a 4-day-old calf and by quantitative PCR in both newborn and fetal calves [55, 56]. Similarly, Mekata et al. [57] reported that calves born to infected dams were PCR-negative during the first 30 days after birth, whereas 88% became infected by 4 months of age, suggesting that vertical transmission may require several months before becoming detectable under low tick challenge conditions.

In Malaysia, Ola-Fadunsin et al. [45] reported no significant difference in infection prevalence between cattle aged ≤1 year (44.5%) and 1–≤2 years (42.22%), whereas significantly higher prevalence was observed in animals aged 2–≤5 years (52.52%) and >5 years (59.64%). Likewise, Selim et al. [39] demonstrated that asymptomatic cattle older than 3 years had a significantly greater prevalence of T. orientalis infection (13.1%; p < 0.003) than cattle aged <1 year (1.6%) or 1–3 years (5.6%). Conversely, cattle residing in endemic regions, including Iraq, may gradually develop partial protective immunity following repeated exposure to infected ticks, resulting in persistent infection without overt clinical disease [14, 20, 58, 59]. Therefore, higher prevalence in older animals may reflect cumulative exposure rather than increased susceptibility to clinical theileriosis.

Effect of sex on T. orientalis infection

Male cattle exhibited significantly higher infection prevalence, OR, and RR than female cattle. In contrast, the prevalence of T. orientalis infection in ticks was not significantly associated with host sex, although ticks collected from male cattle showed a numerically higher risk of infection.

Previous studies have reported inconsistent associations between sex and T. orientalis infection. Selim et al. [39] found no significant difference between female (9.2%) and male (7.5%) cattle, whereas Ola-Fadunsin et al. [45] reported a higher prevalence among females (51.33%) than males (43.89%). The discrepancy between the present findings and previous reports may reflect differences in herd structure, management practices, and animal utilization. In the present study, most female cattle were maintained for dairy production, whereas male cattle were primarily reared for beef production under more extensive grazing conditions, which may increase their exposure to tick vectors.

Phylogenetic relationships of Iraqi T. orientalis isolates

Although T. orientalis has previously been reported in neighboring countries, including Turkey, Iran, and Pakistan, molecular phylogenetic information from Iraq has been lacking despite the country's strategic geographical position linking Asia and the Mediterranean region. In the present study, phylogenetic analysis demonstrated that all cattle-derived T. orientalis isolates clustered closely with the Turkish isolate HQ197736.1. In contrast, tick-derived isolates exhibited the greatest sequence identity with one Chinese isolate (PQ207062.1) and two Turkish isolates (OR211412.1 and HQ197736.1).

These findings suggest regional genetic connectivity and may reflect transboundary movement of infected animals or tick vectors. The greater genetic diversity observed among tick isolates may be explained by ticks' ability to acquire parasites from multiple infected hosts throughout their life cycle, allowing them to harbor a broader range of T. orientalis genotypes than those circulating within individual cattle populations. Recent outbreaks reported in Asia and Australia have been associated with highly pathogenic genotypes, including the Ikeda genotype, which has expanded into previously unaffected regions [6]. Consequently, the present findings contribute important baseline molecular evidence regarding the circulation and potential dissemination of T. orientalis lineages in Iraq. However, the statement regarding "haplotype diversity and network analysis showing shared versus unique haplotypes between cattle and ticks" should be removed because such analyses were not performed in this study.

Utility of the 18S rRNA gene and study limitations

The 18S rRNA gene remains one of the most reliable molecular markers for detecting T. orientalis because of its high copy number and the presence of conserved regions interspersed with hypervariable domains that enable accurate differentiation from other apicomplexan parasites [60–62]. Furthermore, the conserved nature of the 18S rRNA gene facilitates phylogenetic reconstruction and comparison of Theileria isolates worldwide, providing a robust framework for investigating evolutionary relationships and genotype diversity [63–66].

Nevertheless, several limitations should be acknowledged. The study was limited to a single province in Iraq, which may not fully represent the nationwide epidemiology of T. orientalis. In addition, phylogenetic characterization was based solely on the 18S rRNA gene. More discriminatory genetic markers, such as the MPSP gene, ITS regions, mitochondrial genes, or whole-genome sequencing, were not investigated. Consequently, genotype-specific characterization, identification of polymorphic sites, and differentiation of major genotypes, including Ikeda, Chitose, and Buffeli, were beyond the scope of the present study. Future nationwide molecular surveillance incorporating multiple genetic markers and larger sample sizes is therefore warranted to improve understanding of the genetic diversity, transmission dynamics, and epidemiology of T. orientalis in Iraq.